Regeneration of articular cartilage damaged either by disease or injury is a complex problem that remains a significant clinical challenge despite extensive orthopaedic research. Although there are numerous analgesics, therapeutic strategies and surgical procedures developed to address this health concern, most of the therapies are short-lived and non-remedial. The global objective of this project is to repair osteochondral defects with a multilayered construct presenting spatially controlled chondrogenic and osteogenic properties and generated from human mesenchymal stem cells (hMSCs). To this end, the following specific aims are proposed: (1) to develop 3D controlled release systems that deliver plasmid encoded transcription factors at rates and concentrations appropriate for osteogenic and chondrogenic differentiation of MSCs and (2) to evaluate the controlled release systems created in Specific Aim 1 for their potential to generate bi-layered chondrogenic and osteogenic constructs. Osteochondral differentiation of MSCs in 3D scaffolds will be achieved through gene delivery of transcription factors Sox-5, Sox-6, Sox-9 (Sox trio) for chondrogenesis, and Runx-2 for osteogenesis. Each of these plasmids will be complexed with a novel polymeric gene delivery vector developed in our laboratory (a conjugate of branched polyethylenimine and hyaluronic acid) to increase transfection efficiency. Initially, the effect of plasmid concentration and exposure duration on osteochondral differentiation will be examined to identify the appropriate target release profiles. Electrospun, co-axial fiber mesh scaffolds will then be fabricated with the vector-plasmid complexes embedded in the core of the polymer fibers and well-established fabrication variables will be utilized to achieve the desired release profiles. Evaluation of the individual layers for the envisioned final bi-layered construct will be based upon the osteogenic and chondrogenic differentiation of the seeded MSCs and the generation of extracellular matrix similar to the native tissue. The studies will involve hMSCs to evaluate the initial feasibility of the proposed approach for ultimate clinical application in humans, while parallel studies will be conducted using rabbit MSCs to evaluate the appropriateness of the envisioned application of the rabbit animal model in the translational pre-clinical development of this novel approach to osteochondral tissue regeneration. The novelty of this proposal is that it utilizes (a) MSCs to circumvent the traditional problems associated with the limited availability and maintenance of differentiated cell types, (b) transcription factors encoded in plasmids to provide a broader phenotypic induction of MSCs and (c) polymeric scaffolds to provide a three dimensional support lattice for cell proliferation/differentiaton and the controlled release of gene delivery vectors. Although this proposal addresses a specific clinical need, the impact of these studies is not limited to cartilage or even orthopaedic applications. The principles proposed herein provide a means to circumvent the problems associated with the isolation and maintenance of differentiated cells. Transcription factors will potentially facilitate the use of hMSCs in a number of tissue engineering applications. Furthermore, the novel scaffold fabrication technique permits regulated delivery of multiple gene delivery vectors while providing a 3D support for cell growth. These techniques have wide-ranging applications in tissue engineering and can potentially be used to create multilayer scaffolds that mimic the zonal architecture of cartilage and other complex tissues. PUBLIC HEALTH RELEVANCE: This project proposes an innovative technology and approach for creating osteochondral constructs for the repair of damaged articular cartilage and subchondral bone. A combination of developmental biological signals will be utilized in a novel controlled release system to deliver differential signals to induce human mesenchymal stem cells into appropriate osteochondral phenotypes. This system will increase the scope of techniques available to tissue engineers for generating anisotropic tissues with multiple cell types.